Two gamma-interferon-activation sites (GAS) on the promoter of the human intercellular adhesion molecule (ICAM-1) gene are required for induction of transcription by IFN-gamma

We describe the molecular features of the interferon (IFN)-gamma-mediated t ranscription of the human intercellular adhesion molecule (ICAM-1) gene. We identified putative IFN-gamma-activated sites (GAS) distributed throughout a large segment of the ICAM-1 promoter (4.0 kb region). Using computer-ass isted search, these sequences were similar to potential IFN-gamma responsiv e elements that have a core sequence 5'-TTNCNNNAA-3'. In this report we sho w that in the ICAM-1 promoter a GAS site is located at -115 from the transl ation initiation site, and binds with strong affinity to IFN-gamma-activate d Signal Transducers and Activators of Transcription (STAT1) homodimers. Th e same sequence is responsible for the IFN-gamma-mediated transcription of the ICAM-1 gene. Moreover, we present evidence that a more distal GAS eleme nt that maps at -2787 from the translation initiation site, binds IFN-gamma -activated STAT1 dimers with lower affinity. Multimeric copies of such GAS sequence inserted into a tkCAT minimal promoter can drive transcription, de monstrating that the -2787 bp GAS element has an independent functional act ivity upon binding of IFN-gamma-activated STAT1 proteins as documented by i n vitro binding assays. Furthermore, using recombinant ICAM-CAT mutants, we show that, in vivo, the -2787 GAS, but not a mutagenized -2787 GAS site, w hen coupled to the more proximal -115 GAS element, has an additive effect i n enhancing the IFN-gamma-mediated transcription of ICAM-1 promoter. Nevert heless, using a recombinant construct bearing the wild type -2787 GAS eleme nt and a mutagenized -115 GAS element, we could not detect any transcriptio n after transfection of U937 recipient cells, suggesting that the -2787 bp GAS element is not sufficient as such for gene activation, but can cooperat e with its cognate proximal sequence to give full function to the ICAM-1 pr omoter during the IFN-gamma response. Taken together these data provide evi dence that two GAS sites are required for the full potential activity in th e mechanism of ICAM-1 gene activation by IFN-gamma.